A Functional Dried Fruit Matrix Incorporated with Probiotic Strains: Lactobacillus Plantarum and Lactobacillus Kefir

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Focusing on Modern Food Industry (FMFI) Volume 2 Issue 3, August 2013

A Functional Dried Fruit Matrix Incorporated
with Probiotic Strains: Lactobacillus
Plantarum and Lactobacillus Kefir

Rego, A. 1 , Freixo, R. 2 , Silva, J. 3 , Gibbs, P. 4 , Morais, A.M.M.B 5 , Teixeira, P. 6 *

CBQF - Centra de Biotecnologia e Quimica Fina - Laboratorio Associado, Escola Superior de Biotecnologia,
Universidade Catolica Portuguesa/Porto, Rua Dr. Antonio Bernardino Almeida, 4200-072 Porto, Portugal
^ngelamregoOhotmail.com; 2 ricardofreixol@hotmail.com; 3 jlsilva@porto.ucp.pt; 4 pgibbs@porto.ucp.pt;
5 abmorais@porto.ucp.pt; * 6 pcteixeira@porto.ucp.pt

Abstract

The consumption of probiotic functional foods, i.e. processed
foods enriched with microorganisms that confer health
benefits to the host, shows a progressive increase in the last
decade due to changes in habits and trends of consumers
attracted by the benefits of these products. Currently, the
development of fruits and vegetables with probiotic content
is a topic of high interest for the probiotic-food consumers as
these are a popular item perceived as healthy by consumers,
and issues related with lactose intolerance are overcome. The
aim of this research study was to develop a new healthy dry
food that contains a source of probiotic strains providing
some benefits to consumers. Apple was selected as an
experimental food matrix and two different probiotic
Lactobacillus species, L. plantarum and L. kefir, were tested
separately. Samples were taken immediately before and after
the drying process in order to determine the viability of
bacteria adhered to the matrix. Dried apple cubes were
stored in sterile closed glass containers or in sealed bags
vacuum packed and normal atmosphere) at room
temperature and at 4 9 C. The bacterial viability in the dried
product was tested at different storage times. For both
probiotic strains, a decrease of approximately 2 log cycles in
bacterial cell numbers was observed after drying. The
bacterial number in apple cubes at the time of storage at
room temperature and 4 9 C was approximately 1x107 cfu/g.
Both probiotic strains died after one month of storage at
room temperature, while during storage at 4 Q C the cells
remained viable after 3 months, with bacterial number
around 1x106 cfu/g.

Keywords

Probiotic Bacteria; Storage; Survival; Tray Dryer; Immersion;
Vacuum

Introduction

Fruits and vegetables are essential components of the
human diet. Apart from being good sources of
vitamins, minerals, and fibres, these foods are also a

rich source of potentially bioactive compounds
(Palafox et al., 2001). Additionally, the consumption of
fresh fruits as well as functional foods e.g. probiotics,
has increased considerably in recent years, due to the
increasing concern in consumers about food and
health. Combinations of fruits or vegetables with
probiotics (Fito et al., 2001a; Alegre et al., 2010), would
create a better, more convenient, product for
consumers.

Whilst dairy products are the priority of the
development of novel probiotic foods (Puente et al.,
2009), lactose intolerance has been reported and an
alternative to these dairy products is desirable.

The incorporation of probiotic strains in several food
matrixes has been studied maily due to their
therapeutical benefits (Lourens-Hattingh et al., 2001;
Shah et al., 2010). This represents a challenge, since the
viability of the incorporated cells in the food matrix
depends on several factors, such as pH, storage
temperature, oxygen levels, and presence of
competing microorganisms and inhibitors (Mattila-
Sandholm et al., 2002). Vacuum impreganation has
been reported by several authors as a technique to
improve some food characteristics e.g. calcium, iron
salts, pH depressors, antimicrobials, etc (Fito and
Chiralt, 2000; Fito et al., 2000; Fito et al., 2001b)

Probiotics can be added either to fresh foods with high
water activity (aw) or to low aw dry foods. The fresh
foods normally have a shelf-life of a few weeks, like
yogurts, while the shelf-life of dried products is
increased to months, as in the case of milk powder
(Weinbreck et al., 2010).

The principal objective of this research work was to
create a new healthy dried (non-dairy) food containing
a source of probiotic strains bringing some benefits to

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consumers namely in the improvement of the immune
system. Apple "Golden Delicious" was selected as the
food matrix to perform the present research since it is i)
easy to handle; ii) relatively cheap; iii) available in
many countries at any time of the year; iv) highly
nutritious (Obbagy et al., 2009) and v) has a highly
porous matrix, allowing entry of the probiotic
(Krokida et al., 1998). This apple variety is widely
grown and available throughout the year
(Anonymous., 2007).

The specific objectives of the present work were: i) to
establish the conditions for drying apple in a pilot
scale tray dryer; ii) to evaluate the method for
incorporating probiotic LAB (i.e. L. plantarum and L.
kefir) in the apple matrix before the drying process,
and iii) to evaluate the survival rate of the selected
LAB during and after the drying process.

Materials and Methods

Probiotic Bacteria and Growth Conditions

Two Lactobacillus strains, previously described as
probiotic strains (Ouwehand et al., 2002; Vinderola et
al., 2006; Golowczyc et al., 2007, 2009) were selected
for the present study; L. plantarum, which can be found
in fermented foods, and L. kefir, from kefir grains.

These isolates belong to the culture collection of Escola
Superior de Biotecnologia, Universidade Catolica
Portuguesa. Both strains were stored in MRS broth
(Pronadisa, Spain, Madrid) plus glycerol (30% v/v) at -
80 2 C, until use. To prepare the pre-inoculum, a sterile
tube with MRS broth (15 mL) was inoculated with a
pure colony of the selected strain. This suspension was
incubated at 37°C for 24 hours. The pre-inoculum (5
mL, 1% v/v) was added to 500 mL of MRS broth and
incubated at 37°C for 24 hours. This culture was
centrifuged in sterile 50 mL Falcon tubes, (5000 x g at
4°C; Hettich Zentrifugen Rotina 35R, Germany) for 5
minutes followed by two washes of the pelleted cells
by re-suspension and centrifugation, with sterile
Ringer's solution (Merck, Germany, Darmstadt), under
the same conditions. Cell pellets were then re-
suspended in 20 mL of Ringer's solution in each
Falcon tube in order to concentrate the probiotic
suspension before addition to the fruit matrix.

Sample Preparation

The apples used in this study were the variety 'Golden
Delicious' obtained in local markets from the region of
Porto. This variety was chosen because of its sweet

flavour, and the pulp is smooth with a crunchy texture
(Molin, 2001) and as well it does not oxidize very
easily during processing. Apples were washed with
water, peeled and cut into cubes of about 2 cm sides,
using a mold to obtain cubes with the same size and
shape. These apple cubes were immediately immersed
in sterile Ringer's solution to inhibit the oxidation of
the matrix until submersion in the concentrated cell
suspension prepared as described above (500 g of
apple to 1L of sterile Ringer's solution). Cubes
immersed in Ringer's solution were recovered by
filtration using sterile gauze.

Adherence of Probiotic Cells

To promote adherence of probiotic cells into the apple
matrix, two techniques were tested: immersion and
vacuum impregnation (Betoret et al., 2003).

1 ) Immersion

In the immersion technique, the apple cubes were
immersed in the probiotic suspension for one hour.
In order to make this adherence uniform in all
cubes, a gentle agitation over time was applied,
ensuring that all cubes were immersed in the
solution under the same conditions. Afterwards,
the cubes were recovered by filtration under
aseptic conditions using sterile gauze, placed on
trays and then into the dryer (UOP8, Armfield,
United Kingdom).

2 ) Vacuum

A vacuum impregnation technique was also tested
(Maguina et al., 2002). Apple cubes were immersed
in the concentrated cellular suspension, in a bag
suitable for vacuum sealing. A pressure of 50 mbar
for 1.2 seconds was established, with subsequent
sealing of the bag (Multivac A300/52 Vacuum,
United States of America). The bags were opened
and the cubes were removed, also using sterile
gauze, placed on trays that were loaded into the
dryer, where the drying would be accomplished.
Before and after addition to apple cubes,
enumeration of LAB was performed as described
below.

Drying Conditions

A pilot-scale tray dryer (which allows the drying of
wet solid products by flowing hot air over the trays)
was used. Two different temperatures and two
different air velocities were tested. Initially, drying
was performed at room temperature (ca. 20 Q C) and

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with an average speed of air circulation of 0.5 m/s.
These conditions were used to check if the probiotic
bacteria, adhered to apple cubes by the immersion
technique used for the sample preparation, suffered
any decrease in viability, during dehydration. The
duration of this experiment was one week and during
this time several samples were taken and survivors
enumerated. Subsequently, these conditions were
changed. The temperature and speed of air flow were
increased to 40 Q C and 1.5 m/s, respectively. In this
second experiment, drying was faster essentially due
to the increase in temperature. Drying of apple cubes
with adhered LAB, by immersion at normal pressure
or vacuum techniques, occurred in approximately, 27
to 30 hours. Samples were taken during drying
process and survivors were enumerated. The relative
humidity (RH) of the drying air and the aw of the
apple cubes during the drying process were measured
in order to determine the effect of the RH on the
drying of the cubes (Himmelfarb et al., 1962).

Storage Conditions

After drying at 40 Q C, the samples were divided into
two groups, one to test the effect of atmospheric
conditions, and the other to test the effect of
temperature on the viability of the adhered probiotic
bacteria. A portion of the dried apple was stored
under vacuum conditions and the other in sterile
Schott flasks (full flasks with almost no head space)
under normal atmosphere conditions. Apple cubes
that were submitted to vacuum conditions (Multivac
A300/52 Vacuum, United States of America) were
divided into bags and sealed after a pressure of 1
mBar was established. These two groups of samples
(vacuum packed and normal atmosphere) were stored
and then divided in order to determine if storage
temperature had any effect on cell viability. Two
storage temperatures were tested: room temperature
(ca. 20°C) and 4°C.

Bacterial Enumeration

One gram of apples (freshly inoculated) were added to
9 mL of sterile Ringer's solution and mixed in the
stomacher (BagMixer® 400 P, Interscience, France) for
one minute. Then, serial decimal dilutions were
performed and LAB were enumerated by the drop
count technique on MRS agar (Biokar, France,
Beauvais) plates. Colony counting was performed
after incubation at 37 Q C for 24 hours. The same
procedure was followed for samples after drying but
the sample weight was 5 g added to 45 mL of sterile

Ringer's solution.
Results and Discussion

The main objective of the present research was to
create a dry fruit matrix with a high number of viable
probiotic cells (at least > 1x107 cfu/g); therefore it was
crucial: i) to obtain an initial suspension in which the
matrix would be immersed, with a high cell
concentration (-1010 cfu/mL); ii) to assure a high
adherence of the probiotics to the fruit matrix; iii) to
guarantee that after drying, the viability of adhered
cells was still high.

A concentrated probiotic suspension (ca. 1010 cfu/mL)
was produced to allow a high incorporation of the
cells into the food matrix by the two different
techniques, immersion and vacuum. It was established
that one hour of contact would be sufficient to
promote good adherence with a high concentration of
viable cells (ca. 109 cfu/mL; data not shown).

In terms of cell numbers, after one hour immersion, a
difference of one log cycle approximately, was
observed between the initial probiotic suspension and
the immersed apple matrix. According to Fito et al.
(2001a), a vacuum technique would allow the
introduction of controlled quantities of a solution into
the porous structure of fruits. In fact, these authors
described that vaccum impregnation could introduce
into the fruit and vegetables, controlled quantities of a
given solution. However, in the present study, no
differences have been observed between the two
tested techniques, since the same concentration of
viable cells in the apple matrix was achieved at the
same cell suspension concentration by both techniques
(data not shown).

Apple cubes with adhered probiotic bacteria were
dried. After drying, it was observed that cubes that
had been subjected to immersion under vacuum to
promote adherence, presented lower viable numbers
than apple cubes that had been subjected to immersion
at normal pressure conditions (Figs. 1 and 2 for L. kefir
and L. plantarum, respectively). In the course of
comparison of both techniques, a reduction of
approximately 2 log cycles was observed for both
strains for apple samples that were just immersed,
whereas losses were approximately of 4 log cycles for
L. plantarum and near 3 log cycles for L. kefir for apple
cubes that were vacuum treated. Therefore, the
vacuum technique did not confer any advantages in
either increasing the numbers of cells adhered or in

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stability during drying. This was also confirmed in the
study by Alzamora et al. (2005), also using apple cubes
as a food matrix and a different range of vacuum
pressures.

-i

FIG. 1. VIABILITY OF LACTOBACILLUS KEFIR CELLS IN APPLE
CUBES AFTER ADHESION; ""^BY THE IMMERSION
TECHNIQUE: BY THE VACUUM TECHNIQUE. ERROR

BARS INDICATE NO VARIABILITY BETWEEN ASSAYS.

Drying Time (hours)

-5

FIG.2. VIABILITY OF LACTOBACILLUS PLANT ARUM CELLS IN
APPLE CUBES AFTER ADHESION; 1 BY THE IMMERSION
TECHNIQUE; BY THE VACUUM TECHNIQUE. ERROR

BARS INDICATE NO VARIABILITY BETWEEN ASSAYS.

It was also noted that vacuum immersed samples had
a worse visual aspect after drying, with more damage
observed when compared with samples that were not
subjected to vacuum.

Even with the reduction of viable cells (<2 log cfu/g)
observed in the apple cubes inoculated by simple
immersion, the lactobacilli continued to be present in
large numbers, (ca. 107 cfu/g), even at the end of the
drying process. Much greater reductions of viable cells
(ca. 3-4 log cfu/g) were noted for both lactobacilli in
apple cubes inoculated by vacuum immersion (Figs. 1
and 2).

When these results were compared with Betoret et al.
(2003), some differences in the final concentrations of
incorporated cells in the final product were observed.
To promote adherence (Betoret et al., 2003), fruit juices
or even milk inoculated with probiotic bacteria were
used. These suspensions were then put in contact with

the apple slices to promote the incorporation of the
bacteria in the matrix. This step led to a better
incorporation of the probiotic bacteria into the apple
slices, when compared to the results obtained in the
current study. The use of juice or milk seemed to
confer some protection to the probiotic cells, making
them more resistant to drying. The pressure used for
vacuum immersion was the same, but Betoret et al.
(2003) applied it for a longer period. This may have
had some advantages for the incorporation of the cells,
since in the currently reported study, vacuum was
applied for only 1.2 seconds at 50 mBar instead the 10
minutes used by Betoret et al. (2003) to promote
adherence. These differences in time could lead to
different adherences of the cells to the matrix.

Dried apple cubes incorporated with the two probiotic
LAB by both methods, were stored at room
temperature (ca. 20°C) in closed glass bottles in the
dark for up to 25 days. After 24 days of storage, viable
cells of L. kefir, incorporated by either method, had
decreased by ca. 1 log cfu/g (Fig.3).

Drying Time (hours)

-0,5 '

-1 -

-1,5 -

-2 -

1

BC

-2,5 -

-!

-3 -

-3,5 -

-4 -

-4,5 -

FIG.3. VIABILITY OF LACTOBACILLUS PLANTARUM CELLS IN
APPLE CUBES DURING DRYING TIME;. ™^ IMMERSION
TECHNIQUE TO PROMOTE ADHERENCE; - VACUUM
TECHNIQUE TO PROMOTE ADHERENCE. ERROR BARS
INDICATE NO VARIABILITY BETWEEN ASSAYS.

After 20 days of storage, L. plantarum viable cells
incorporated into apple by immersion, had decreased
by ca. 0.5 log cfu/g, but cells incorporated by the
vacuum technique had decreased by ca. 1.5 log cfu/g
(Fig.4). In an attempt to minimize the loss of viability
of L. plantarum, vacuum infused apple cubes were also
stored under vacuum, since it was possible that air -
oxygen — was deleterious for cell survival. After just
eight days of storage at ambient temperature, vacuum-
stored cells had lost ca. 4 log cfu/g, and could not be
recovered thereafter; cells could be recovered after 25
days storage in normal atmosphere, although with a

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loss of viability of ca. 2 log cfu/g (Fig. 5).

i

().-

g

^05

-1
-1,5
-2

Storage time (Days)

10 15

25 30

FIG.4. VIABILITY OF LACTOBACILLUS KEFIR CELLS IN APPLE
CUBES DURING DRYING TIME;. ™^ IMMERSION
TECHNIQUE TO PROMOTE ADHERENCE; VACUUM
TECHNIQUE TO PROMOTE ADHERENCE. ERROR BARS
INDICATE NO VARIABILITY BETWEEN ASSAYS.

Sloraje lime (Days)

FIG.5. EFFECT OF VACUUM TECHNIQUE USED TO PROMOTE
ADHESION OF LACTOBACILLUS PLANT ARUM, ON ITS
SURVIVAL DURING STORAGE AT ROOM TEMPERATURE.
™^ STORAGE AT NORMAL ATMOSPHERE; STORAGE
UNDER VACUUM.

Over several replicated experiments with both LAB
incorporated into apple cubes by immersion, and
drying by air at 40°C, the cell viability losses by drying,
were between 1.5 and ca 3 log cfu/g (data not shown).
A possible reason for the differences recorded is that
the RH of the heated air was not controlled, thus
giving different rates of drying, and with low RH
there may have been rapid surface dehydration and
prolonged dehydration of the interior.

Golowczyc et al., (2009) observed, using the same
strains used in this study, that L. plantarum was more
resistant to high temperatures than L. kefir. As
reported by other authors, these strains of Lactobacillus
are capable of growth in this range of temperature (De
Vos, 2009), so cell death is probably the decrease in
water content, leading to shrinkage of the cell
membrane and to the death of cells.

After one month of storage at room temperature, the
viability of cells in apple slices decreased by 4 log
cycles. However, storage at 4°C resulted in a loss of

viability of only 1 log cfu/g even after 65 days of
storage (Fig. 6). As in the study of Alzamora et. al.
(2005), samples that were incorporated with probiotics,
only lost one log cycle of viability during storage at 4
Q C and could remain stable for long periods at that
temperature. So, it was possible to conclude that, for
the conditions investigated, storage in normal
atmosphere at 4 Q C was the best way to preserve
probiotic cell viability in dried apple cubes.

Storage Ti me (Days}

FIG.6. SURVIVAL OF CELLS IN DRIED APPLE CUBES DURING
STORAGE. ""^ LACTOBACILLUS PLANTARUM SURVIVAL
AT ROOM TEMPERATURE; LACTOBACILLUS
PLANTARUM SURVIVAL AT 4 °C; LACTOBACILLUS
KEFIR SURVIVAL AT ROOM TEMPERATURE;
LACTOBACILLUS KEFIR SURVIVAL AT 4 Q C.

After drying the apple matrix, the cubes were stored
for at least one month, to check the cell viability and
shelf life under storage conditions. Several factors
could influence the quality of the product, including
temperature, moisture content, and atmosphere
composition in which the product is stored
(Anonymous, 2001).

ACKNOWLEDGMENT

This work was supported by National Funds from
FCT - Fundacao para a Ciencia e a Tecnologia through
project PEst-OE/EQB/LA0016/2011. Financial support
for author Joana Silva was provided by Postdoctoral
fellowship SFRH/BPD/35392/2007 (FCT)

REFERENCES

Anonymous. Maca. Ministerio da Agriculture, do

Desenvolvimento Rural e das Pescas, Gabinete de

Planeamento e Politicas 2007, pp. 1-17.
Anonymous, FDA. Evaluation and Definition of Potentially

Hazardous Foods. U.S. Department of Health & human

Services, FDA, 2001.
Alegre, I., Vinas, I., Usall, J., Anguera, M., and Abadias, M.

Microbiological and physicochemical quality of fresh-cut

142

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www.fmfi-journal.org

apple enriched with the probiotic strain Lactobacillus
rhamnosus GG. Food Microbiology 28 (2010): 59-66.

Alzamora, S.M. et al., Novel functional foods from vegetable
matrices impregnated with biologically active
compounds. Journal of Food Engineering 67 (2005): 205-
214.

Betoret, N. et al., Development of probiotic-enriched dried
fruits by vacuum impregnation. Journal of Food
Engineering 56 (2003): 273-277.

De Vos et al., Order II. Lactobacillales ord. nov. In: Bergey's
Manual of Systematic Bacteriology: The firmicutes
(Second Edition) pp. 464-510, 2009.

Fito, P. et al., Vacuum impregnation and osmotic
dehydration in matrix engineering Application in
functional fresh food development. Journal of Food
Engineering 49 (2001a), 175-183.

Fito, P. et al., Vacuum impregnation for development of new
dehydrated products. Journal of Food Engineering 49
(2001b), 297-302.

Fito, P. and Chiralt, A. Vacuum impregnation of plant
tissues. In Alzamora, S.M. et al. (Eds.), Minimal
processed fruits and vegetables (pp. 189-201). Maryland:
Aspen Publishers (2000).

Golowczyc, M.A., Silva, J., Abraham, A.G., De Antoni, G.L.
and Teixeira, P. Preservation of probiotic strains isolated
from kefir by spray drying. Letters in Applied
Microbiology 50 (2009): 7-12.

Golowczyc, M.A.; Mobili, P.; Garrote, G.L.; Abraham, A.G.
and Antoni, A.G. Protective action of Lactobacillus kefir
carrying S-layer protein against Salmonella enterica
serovar Enteritidis. International Journal of Food
Microbiology 118 (2007): 264-273.

Himmelfarb, P., El-Bisi, H.M., Read,R.B. and Litsky,W. Effect
of relative humidity on the bactericidal activity of
propylene oxide vapor. Institute of Agricultural and
Industrial Microbiology, University of Massachusetts 10
(1962): 431-435.

Krokida, M.K., Karathanos, V.T. and Maroulis, Z.B. Effect of
freeze-drying conditions on shrinkage and porosity of
dehydrated agricultural products. Journal of Food
Engineering 35 (1998): 369-380.

Lourens-Hattingh, A. and Viljoen, B.C. Yogurt as probiotic

carrier food. International Dairy Journal 11 (2001): 1-17.
Maguina, G. et al, Incoporation of Bifidobacterium spp by

hydrodynamic mechanism in a porous fruit matrix.

Annual Meeting and Food Expo - Anaheim, California,

2002.

Mattila-Sandholm et al., Technological challenges for future
probiotic foods. International Dairy Journal 12 (2002):
173-182.

Molin, G. Probiotics in foods not containing milk or milk
constituents, with special reference to Lactobacillus
plantarum 299v. American Journal of Clinical Nutrition
73 (2001):380 - 385.

Obbagy, J.E. and Rolls, B.J. The effect of fruit in different
forms on energy intake and satiety at a meal. Appetite 52
(2009): 416-422.

Ouwehand, A.C.; Salminen, S. and Isolauri, E. Probiotics: an
overview of beneficial effects. Antonie van Leeuwenhoek
82 (2002): 279-289.

Palafox, H.C., Zavala, J. and Gonzalez, G.A. The role of
dietary fiber in the bioaccessibility and bioavailability of
fruit and vegetable antioxidants. Journal of Food Science
76 (2011): 6-15.

Puente, L.D., Betoret, N.V., Cortes, M.R. Evolution of
probiotic content and color of apples impregnated with
lactic acid bacteria. Vitae, Revista de la Facultad de
Quimica Farmaceutica 16 (2009): 297-303.

Shah, N.P., Ding, W.K., Fallourd, M.J. and Leyer, G.
Improving the stability of probiotic bacteria in model
fruit juices using vitamins and antioxidants. Journal of
Food Science 75 (2010): 278-282.

Vinderola, G., Perdigon, G., Duarte, J., Farnworth, E. and
Matar, C. Effects of the oral administration of the
exopolysaccharide produced by Lactobacillus
kefir anofaciens on the gut mucosal immunity. Cytokine 36
(2006): 254-260.

Weinbreck, F., Bodnar, I., Marco, M.L. Can encapsulation
lengthen the shelf-life of probiotic bacteria in dry
products? International Journal of Food Microbiology
136 (2010): 364-367.

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